Antibiotic X-1092

ABSTRACT

The invention relates to a new and useful antibacterial substance which is of the formula ##EQU1## and to processes for its production and recovery. The invention embraces this antibacterial agent and its salts as crude concentrates, as purified solids and in pure crystalline forms. This antibiotic of the Formula I is effective in inhibiting the growth of gram positive bacteria. The compound of the Formula I is prepared by cultivating a strain of Streptomyces sp. X-1092 in an aqueous carbohydrate solution containing a nitrogenous nutrient under submerged aerobic conditions until substantial activity versus Gram-positive bacteria is imparted to said solution and then recovering said compound of the Formula I from said solution.

This is a division of application Ser. No. 504,598 filed Sept. 9, l974, now U.S. Pat. No. 3,939,139.

DETAILED DESCRIPTION OF THE INVENTION

There is provided, according to the present invention, an antibiotic substance effective in inhibiting the growth of Gram-positive bacteria which is of the Formula I. Chemically, this substance is known as 1-(S)-hydroxy-2-(S,S)-valylamido-cyclobutane-1-acetic acid.

There is further provided according to the present invention, a process for the production of such antibiotic substance of the Formula I which comprises cultivation of a strain of Streptomyces sp, X-1092 in an aqueous carbohydrate solution containing a nitrogenous nutrient under submerged aerobic conditions until substantial activity versus gram-positive bacteria is imparted to said solution and then recovering said compound of the Formula I from said solution.

The organism producing the antibiotic of the present invention is a new species designated Streptomyces sp. X-1092. A culture of the living organism, given the laboratory designation X-1092, has been deposited in the U.S. Department of Agriculture, Agriculture Research Service, NRRL, Peoria, Ill., and added to its permanent collection of microorganisms as NRRL 8047.

The representative strain of Streptomyces sp. X-1092 has the following characteristics.

On solid media, this organism grows in a fashion that is characteristic for members of the genus Streptomyces: abundant mycelial growth penetrates into the agar, and the aerial mycelium breaks up in characteristic chains of spores while there is no fragmentation of the immersed mycelium. The aerial sporulation system belongs to the type retinaculum-apertum (RA), with loose spirals, loops and hooks of small diameter.

The formulation of the media used in the description of the growth characteristics, is described below.

Medium 1: "Fermentation medium": Difco Bacto Thermoactinomyces fermentation medium to which 1.5% agar was added.

Medium 2: BBL(Baltimore Biological Laboratory) Czapek-Dox, to which 1.5% agar was added.

Medium 3: Difco Bacto potato dextrose agar.

Medium 4: "Y+M medium": yeast extract, 0.4%; malt extract, 1.0%; dextrose, 0.4%; agar, 2%; pH 7.3.

Medium 5: "Oatmeal-glucose medium": Gerber's oatmeal, 5%; dextrose, 2%; agar 2% in tap water; pH 7.0.

Medium 6: "Tomato paste medium": dextrose, 1% K₂ HPO₄, 0.1%; tomato paste, 2%; Wilson's Medopeptone, 0.1%; CaCO₃ ;0.2%; agar, 1.5% in tap water; pH 6.8-7.3.

Medium 7: "Tomato-soy medium": same as medium 6, to which 1% soyalose (Central Soya Co.) has been added.

Medium 8: "Pablum medium": 6% Pablum mixed cereal in cheesecloth bag dipped in and out of boiling tap water for 2 or 3 minutes. The water lost by evaporation is replaced, and agar is added to 1.5%.

Medium 9: "Tomato-oatmeal" medium": baby oatmeal (Gerber's), 2%; tomato paste, 2%; agar, 2%, in tap water; pH 6.8-7.3.

Medium 10: "Yeast extract medium": yeast extract, 1%; dextrose, 1%; agar, 1.5%, in tap water; pH 6.8.

Medium 11: "Glucose-asparagine medium": dextrose, 1%; asparagine, 0.05%; K₂ HPO₄, 0.05%; agar, 1.5%; pH 6.8.

Medium 12: "Glycerol-asparagine medium": glycerol, 1%; asparagine, 0.1% K₂ HPO₄, 0.1%; agar, 2% in tap water; pH 7.0.

Medium 13: "Starch-casein medium": soluble starch, 1%; casein, 0.1%; K₂ HPO₄, 0.05%; MgSO₄, 0.05%; agar, 1.5%; pH 7.4.

Medium 14: "Emerson's medium": beef extract, 0.4%; peptone, 0.4%; Nacl, 0.25%; yeast extract, 0.1%; dextrose, 1%; agar, 2%; pH 7.0.

Medium 15: "Bennett's medium": yeast extract, 0.1%; beef extract, 0.1%; N-Z-Amine A (casein hydrolysate from Sheffield Inc.) 0.2%; dextrose, 1%; agar, 1.8%; pH 7.3.

Medium 16: "Amidex medium": Amidex (Corn Products Co., Decatur, Ill.) 1%; N-Z-Amine A, 0.2%; beef extract, 0.1%; yeast extract, 0.1%; CaCl₂, 2H₂ O, 0.0014%; agar, 2%; pH 7.3.

Medium 17: "Sporulation medium" (ATCC medium number 5): yeast extract, 0.1%; beef extract, 0.1%; tryptose 0.2%; glucose, 1%; FeSO₄, trace; agar, 1.5%; pH 7.2.

Media 18, 19, 20, 21, 22, 23 are, respectively, media 2 through 7 as described by Shirling, E. G., and Gottlieb, D., Methods for characterization of Streptomyces species, International J. of Systematic Bacteroil., 16, 313-340, 1966.

Unless otherwise indicated, the observations reported have been performed after a period of incubation of 14 days at 28° C. The characteristics of growth in these solid media is summarized in the Table below. Colors of upper surface and reverse are named according to Ridgway's Color Standards and Color Nomenclature, Washington, D.C., 1912.

    __________________________________________________________________________         Characteristics of                                                                          Color of upper surface                                                                      Color of reverse of                              Medium                                                                             aerial growth                                                                               of colonies  colonies                                         __________________________________________________________________________      1  abundant; wrinkled;                                                                         pallid quaker drab                                                                          dirty cream buff                                     good sporulation                                                                            turning to medium deep                                                                      center turning to                                                 mouse gray toward the                                                                       dark olive and pale                                               edges; whitish edges                                                                        olive buff toward                                                              the edges                                         2  fair; flat, thin                                                                            whitish      whitish                                              sporulation                                                                 3  almost none                                                                 4  abundant; wrinkled;                                                                         pale mouse gray                                                                             dirty olive ocher                                    good sporulation;                                                              medium becomes                                                                 yellow; droplets of                                                            yellow exudate on                                                              colonies                                                                    5  none                                                                        6  abundant; wrinkled;                                                                         light mouse gray,                                                                           honey yellow center                                  granulated;  with whitish edges                                                                          and light brownish                                   sporulation thicker       olive toward the                                     at the edges; yellow      edges, with                                          pigment diffuses          mustard yellow                                       into medium               edges                                             7  abundant; wrinkled;                                                                         pale mouse gray; off-                                                                       pale mouse gray                                      with good    white edges  center, mouse                                        sporulation; medium       gray toward the                                      turns light yellow        edges, and off-                                                                white edges                                       8  abundant; wrinkled;                                                                         pale mouse gray and                                                                         mustard yellow                                       granulated, good                                                                            off-white edges                                                                             center turning                                       sporulation               buffy citrine at                                                               edges                                             9  abundant; wrinkled;                                                                         light mouse gray with                                                                       honey yellow center                                  granulated; good                                                                            off-white edges; later                                                                      turning light                                        sporulation; traces                                                                         mouse gray with gray                                                                        brownish olive                                       of light yellow                                                                             edges        toward the edges                                     pigmentation in                                                                the medium                                                                 10  abundant; wrinkled;                                                                         light mouse gray with                                                                       antimony yellow,                                     good sporulation;                                                                           off-white edges which                                                                       and in parts,                                        antimony-yellow                                                                             in time become pallid                                                                       ochraceous tawny                                     exudate; yellow                                                                             mouse gray                                                        diffusible pigment                                                         11  good; wrinkled;                                                                             barium yellow vegeta-                                                                       citron yellow                                        scanty sporulation                                                                          tive mycelium                                                     off-white in color                                                         12  abundant; thin                                                                              cream color vegeta-                                                                         primuline yellow                                     sporulation, white                                                                          tive mycelium; later,                                             in color; colonies                                                                          pallid mouse gray to                                              granular, cracking                                                                          light mouse gray, with                                            with age; light                                                                             off-white edges                                                   yellow diffusible                                                              pigment                                                                    13  abundant; light                                                                             vegetative mycelium                                                                         barium yellow                                        sporulation; yellow                                                                         primrose yellow;                                                  pigment produced;                                                                           sporulated areas pallid                                           clearing around the                                                                         mouse gray with off-                                              colonies (hydrolysis                                                                        white edges                                                       of starch and/or casein)                                                   14  abundant; fairly                                                                            vegetative mycelium                                                                         dirty mustard yellow                                 abundant sporulation                                                                        Naples yellow; spore                                                                        center turning dirty                                              areas pallid mouse                                                                          buffy brown and Naples                                            gray turning off-                                                                           yellow at the edges                                               white at the edges                                            15  abundant; and good                                                                          pale mouse gray; edges                                                                      wax yellow center                                    sporulation, which                                                                          light mouse gray; off-                                                                      turning into dirty                                   is thinner at the                                                                           white edges  olive citrine close                                  center of colonies;       to the edges                                         drops of exudate on                                                            colonies; light                                                                yellow pigment                                                                 diffuses into medium                                                       16  good growth and                                                                             vegetative mycelium                                                                         mustard yellow center                                sporulation  chamois; sporulation                                                                        turning to antimony                                               off-white with mouse                                                                        yellow toward the                                                 gray patches;                                                                               edges                                                             vegetative mycelium at                                                         edges of colonies                                                              becomes yellow ocher                                          17  abundant; poor                                                                              vegetative mycelium                                                                         olive ocher center                                   sporulation; honey yellow;                                                                               turning to honey                                     light yellow sporulated areas light                                                                      yellow toward edges                                  pigment diffusing                                                                           mouse gray with off-                                              into medium  white edges                                                   18  abundant; wrinkled;                                                                         mouse gray with few                                                                         fuscous; paler indenta-                              with surface cracks;                                                                        pale gray patches                                                                           tions immersed into                                  good sporulation;         the agar                                             light yellow                                                                   diffusible pigment                                                         19  good, flat, with                                                                            vegetative mycelium                                                                         cream buff                                           ruffled edges;                                                                              cream buff; sporulated                                            thin sporulation                                                                            areas pallid mouse gray                                       20  abundant; smooth,                                                                           mouse gray at center                                                                        deep grayish olive                                   with ruffled edges;                                                                         and pale mouse gray                                                                         center changing to                                   good sporulation;                                                                           near edges; off-white                                                                       chamois and cream                                    lumps near the                                                                              edges        buff toward edges                                    center of colonies;                                                            very little clear-                                                             ing around the                                                                 colonies                                                                   21  abundant; with                                                                              mouse gray   mouse gray                                           good sporulation                                                           22  fair; no sporulation;                                                                       Naples yellow                                                                               mustard yellow                                       light yellow pigment                                                           diffuses into the                                                              medium                                                                     __________________________________________________________________________

Unless otherwise indicated, the observations reported have been performed after a period of incubation of 14 days at 28° C. The characteristics of growth in these solid media are summarized in the Table above. Colors of upper surface and reverse are named according to Ridgway's Color Standards and Color Nomenclature, Washington, D.C., 1912.

The species Streptomyces X-1092 described herein includes all strains of streptomyces which form a compound of the Formula I and which cannot be definitely differentiated from the strain NRRL 8047 and its subcultures including mutants and variants. The compound of the Formula I is identified herein and after this identification is known, it is easy to differentiate the strains producing a compound of the Formula I from others.

Streptomyces sp. X-1092, when grown under suitable conditions, produces a compound of the Formula I. A fermentation broth containing Streptomyces sp. X-1092 is prepared by inoculating spores or mycelia of the compound of the Formula I -- producing organism into a suitable medium and then cultivating under aerobic conditions. For the production of a compound of the Formula I, cultivation on a solid medium is possible but for production in large quantities cultivation in a liquid medium is preferable. The temperature of the cultivation may be varied over a wide range, 20°-35° C, within which the organism may grow but a temperature of 26°-30° C and a substantially neutral pH is preferred. In the submerged aerobic fermentation of the organism for the production of a compound of the Formula I, the medium may contain as the source for carbon, a commercially available glyceride oil or a carbohydrate such as glycerol, glucose, maltose, lactose, dextrin, starch, etc. in pure or crude states and as the source of nitrogen, an organic material such as soybean meal, distillers solubles, peanut meal, cotton seed meal, meat extract, peptone, fish meal, yeast extract, corn steep liquor, etc. and when desired inorganic sources of nitrogen such as nitrates and ammonium salts and mineral salts such as ammonium sulfate, magnesium sulfate and the like. It also may contain sodium chloride, potassium chloride, potassium phosphate and the like and buffering agents such as sodium citrate, calcium carbonate or phosphates and trace amounts of heavy metal salts. In aerated submerged culturing procedures, an anti-foam agent such as liquid paraffin, fatty oils or silicone compounds is used. More than one kind of carbon source, nitrogen source or anti-foam source may be used for production of a compound of the Formula I. Generally the cultivation is continued until at least several hundred mcg/ml of a compound of the Formula I has accumulated in the medium.

The following Examples will serve to illustrate this invention without limiting it thereto.

EXAMPLE 1

Spores of Streptomyces sp. X-1092, were added to 6-liter Erlenmeyer flasks containing 2 liters of Trypticase soy broth (Baltimore Biological Laboratories). The flasks were incubated at 28° for 76 hours on a rotary shaker [240 rpm with a 2 inch stroke]. Four liters of inoculum was added to 200 liters of fermentation medium containing (in g/liter): K₂ HPO₄, 7.0; KH₂ PO₄, 3.0; (NH₄)₂ SO₄, 1.0; sodium citrate, 0.5; MgSO₄. 7H₂ 0, 0.1; and D-glucose, 2.0 (autoclaved separately). The culture was incubated at 20° in a 380-liter fermentor, aerated at 113 liters per minute and agitated at 260 rpm. Silicone anti-form (Dow Corning AF) was added as needed to control frothing. After 66 hours of incubation, the fermentation broth was filtered through infusorial earth.

EXAMPLE 2

The clarified broth from two 200 liter fermentations was applied to 50 liters Dowex 50WX-4 resin, styrene-divinyl benzene-sulfonic acid ion exchange resin Ca 50- 100 mesh, in the H+ form. After washing with 200 liters distilled water, the resin was eluted with 400 liters 5% aqueous pyridine solution. The eluate which contained 33 g solids was evaporated under reduced pressure to 2 liters, the pH adjusted to 2.5 by addition of 5N HCl and the solution applied to a column (70 cm ht) containing 2.5 liters Bio-Rad AG50WX-4 resin, styrene-divinyl benzene-sulfonic acid ion exchange resin, 100- 200 mesh, in the Na+ form, which had been equilibrated with 0.2M sodium phosphate-citrate buffer, pH 4.2. The resin was then eluted with the same buffer and the activity was obtained at an elution volume of 11-13 liters. This fraction was desalted by readsorption of the antimetabolite onto 1.2 liters Bio-Rad AG50WX- 4 resin, 50- 100 mesh, in the H+ form followed by elution with 10% aqueous pyridine solution. The eluate was evaporated under reduced pressure to a small volume, the concentrate was treated with charcoal, the filtrate from the charcoal step was again evaporated and 1-(S)-hydroxy-2-(S,S)-valylamido-cyclobutane-1-acetic acid was crystallized from ethanol-water (9-1):m.p. 247°-250°; [α]_(D) ²⁵ + 8.4 (c 1, H₂ O), -31.2 (c 1, 5N HCl); pK₁ 3.8, pK₂ 7.5; ir (Kbr disk) 3365 and 3240 (OH and amide NH), 1660 and 1520 (sec amide), 1615 and 1390 cm⁻ ¹ (carboxylate); nmr (D₂ O, 20 mg, ext TMS), δ 4.61 ##STR1## 4.23 (d,1 J = 6Hz, 2.92 (3.18 with DCl) ##STR2## 2.30-2.80 (m,5), 1.47 (d,6, J = 6.5Hz, ##STR3## Anal. calcd for C₁₁ H₂₀ N₂ O₄ : C 54.03, H 8.25 N 11.47. Found: C 54.11, H 8.40, N 11.57.

EXAMPLE 3

As indicated above the compound of the Formula I and its salts are active against gram positive bacteria. Listed in Table 1 which follows are the inhibition zone diameters (mm) for several illustrative organisms as determined by agar-diffusion testing. As is apparent from the foregoing properties, the antibiotic of the Formula I is useful for suppressing the growth of gram positive organisms.

The antimicrobial spectrum of 1-(s)-hydroxy-2-(S,S)-valylamido-cyclo-butane-1-acetic acid was measured in a chemically defined medium^(a) by the paper disc agar-diffusion technique. The results are given in Table 1. The activity was limited to gram positive organism.

                                      Table 1                                      __________________________________________________________________________     Antimicrobial Spectrum                                                         Test Organism      Inhibition zone diameter (mm)                               __________________________________________________________________________     Bacillus cereus ATCC-6464                                                                         60                                                          Bacillus sp. ATCC-27860                                                                           25                                                          Bacillus subtilis NRRL-558                                                                        34                                                          Streptomyces cellulosae ATCC-3313                                                                 34                                                          Micrococcus glutamicus ATCC-13761.sup.b                                                           20                                                          Escherichia coli B  0                                                          Pseudomonas ovalis NRRL-22                                                                         0                                                          Candida albicans NRRL-477.sup.b                                                                    0                                                          Pullularia pullulans QM-279c                                                                       0                                                          __________________________________________________________________________

a. Paper-disc agar-diffusion assays were performed with 12.7 mm discs each containing 12 mμ of a compound of the Formula I in Davis Minimal Agar.

b. Biotin was added to the medium at 100 g liter to insure ample growth of these test organisms.

The compound of the Formula I also evidenced anti-metabolite activity. This activity was determined by counter diffusion methods which have been described in the literature in Journal of Antibiotics, Volume 27, pp. 229-233, 1974. against Streptomyces cellulosae. The activity of the compound of the Formula I was non-competitively reversed by the addition of either L-cysteine or L-cystine to the medium. Partial reversals were observed with either L-methionine or D,L-homocysteine. Other common amino acids, nucleosides and water soluble vitamins did not reverse the activity of a compound of the Formula I. In addition glutathione and dithiothreitol did not reverse the inhibition, so it can be concluded that the reversal by cysteine is not due to chemical inactivation by thiols.

Only slight reversal of inhibition against the three species of Bacilli and Micrococcus glutamicus was observed with L-methionine, L-cystine and L-cysteine; no reversal was observed with other common amino acids mucleosides and/or water soluble vitamins.

As is indicated above, the compound of the Formula I is prepared under submerged aerobic conditions. Preferably submerged fermentation in tanks is used for production of substantial quantities of the X-1092 antibiotic in accordance with conventional procedures. Small quantities of antibiotic are obtained by shake-flask culture. As is customary in aerobic submerged culture processes, sterile air is blown through the culture medium. For efficient growth of the organism and production of the antibiotic of the Formula I, the volume of air employed in the production is above 0.1 volume of air per minute per volume of culture medium. Optimum growth occurs when the volume of air employed is between 0.6 and one volume of air per minute per volume of culture production medium. The production of antibiotics can be followed during the fermentation by testing samples of the broth for antibiotic activity against organisms known to be sensitive to the antibiotic. The bioassay is conveniently effected by paper disc assay on agar plates.

As is the custom, maximum antibiotic production occurs within 2-6 days in either large tank or shake-flask fermentation. Commonly maximum production of antibiotic activity is realized within 5-6 days.

Following its production under submerged aerobic conditions the compound of the Formula I can be recovered from the fermentation broth by methods commonly employed in the fermentation art. The antibiotic activity produced during fermentation of a compound of the Formula I-producing organism occurs in the antibiotic broth. Accordingly, isolation techniques employed in the production of such antibiotics are designed to permit maximum recovery of the antibiotic from the broth. Thus, for example, mycelia and undissolved solids are removed from the fermentation broth by conventional means such as filtration and the antibiotic of the Formula I is recovered from the filtered broth by techniques such as ion exchange or adsorption.

The compound of the Formula I having the basic amino group and the acid COOH group can form salts with both acids and bases. It is easily soluble in alcaline solutions such as aqueous solutions of alkaline metal and alkaline earth metal hydroxides. Thus, solutions of a compound of the Formula I in aqueous sodium hydroxide or calcium hydroxide forms the sodium or calcium salt respectively which can be isolated by conventional procedures. In like manner are formed salts of organic bases such as amine salts and the like. Similarly, the compound of the Formula I can form salts with acidic substances and such substances can be prepared by conventional techniques with such pharmaceutically acceptable acids as hydrochloric acid, hydrobromic acid, sulfuric acid and the like. All that is required of the salt is that it provide a pharmaceutically acceptable salt of a compound of the Formula I.

As is indicated above, compounds of the Formula I and its salts possess the property of adversely affecting the growth of certain Gram-positive bacteria. It is useful in wash solutions for sanitary purposes as in the washing of hands and the cleaning of equipment, floors or furnishings of contaminated rooms or laboratories; It is useful also for suppressing the growth of sensitive organisms in plate assays and other microbiological media. 

We claim:
 1. The process for the production of an antibiotic substance of the formula ##EQU2## which comprises cultivating a strain of Streptomyces sp. X-1092 deposited as NRRL 8047 in an aqueous carbohydrate solution containing a nitrogenous nutrient under submerged earobic conditions until activity versus Gram-positive bacteria is imparted to said solution and then recovering said antibiotic of the formula I from said solution. 